Phospholipase C;1 Is Required for Metastasis Development and Progression

Research Article

Gianluca Sala,1 Francesco Dituri,1 Claudio Raimondi,1 Sara Previdi,2 Tania Maffucci,1 Marco Mazzoletti,2 Cosmo Rossi,3 Manuela Iezzi,3 Rossano Lattanzio,3 Mauro Piantelli,3 Stefano Iacobelli,3 Massimo Broggini,2 and Marco Falasca1

1Inositide Signalling Group, Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London, United Kingdom; 2Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; and 3Center of Excellence on Aging, G. d’Annunzio University Foundation, Chieti, Italy

Abstract critical role in both cytoskeletal changes and migration associated g Cell motility and invasion play an essential role in the with the metastatic process (5, 6). Activation of PLC 1 can occur in development of metastasis. Evidence suggests that the enzyme response to either growth factors or integrin receptors–dependent phospholipase C;1(PLC;1) may be involved in tumor pathways and induces hydrolysis of phosphatidylinositol-4,5- progression and possibly development of metastasis. In this bisphosphate (PtdIns-4,5-P2) to form the second messengers study, we show that down-regulation of PLC;1 expression diacylglycerol and inositol-1,4,5-trisphosphate, which in turn g severely impairs activation of the small GTP-binding protein activate a number of signaling molecules (7, 8). PLC 1 has been Rac and cell invasion in breast cancer cell lines and U87 shown to play a critical role in cell migration, invasion, and g in vitro. Experimental metastasis assays in nude mice show spreading (9–12). Indeed, PLC 1 is highly expressed in several tumors, such as breast carcinomas, and in highly metastatic that inducible knockdown of PLC;1 strongly inhibits development of MDA-MB-231–derived lung metastasis and reverts colorectal tumor cell lines (13, 14). In particular, we showed g metastasis formation. In addition, analysis of 60 breast cancer previously that phosphoinositide 3-kinase (PI3K)–mediated PLC 1 patients’ tissues revealed an increase of PLC;1 expression activation is required for epidermal growth factor (EGF)–induced g in metastasis compared with the primary tumor in 50% of migration of breast cancer cells (15) but the mechanism of PLC 1- tissues analyzed. These data showa critical role of PLC; 1in dependent cell migration and metastasis was not clearly defined. g the metastatic potential of cancer cells, and they further Although previous studies provide evidences that PLC could be indicate that PLC;1 inhibition has a therapeutic potential in involved in carcinoma invasion, it should be noted that this the treatment of metastasis dissemination. [Cancer Res observation was based on the use of generic PLC chemical g 2008;68(24):10187–96] inhibitor (11) or dominant-negative PLC (16). Here, we investigate the role of PLCg1 in cell invasion and metastasis using different approaches to modulate PLCg1 expression in highly invasive Introduction cancer cell lines. Our results show that PLCg1 is required for breast Metastasis, the ability of cancer cells to spread from a primary cancer cell invasion and Rac1 activation. These data reveal a site and form tumors at distant sites, is the main cause of death functional link between PLCg1 and Rac1 that provides insight into associated with cancer. Several steps regulate the development of processes regulating cell invasion. Furthermore, we show that metastasis (local invasion, intravasation, survival, extravasation, down-regulation of PLCg1 expression inhibits the human breast initiation, and maintenance of micrometastases at distant sites and cancer cell line MDA-MB-231–derived lung metastasis develop- vascularization of the resulting tumors). Cell motility and invasion ment in in vivo mice model. According to this, immunohistochem- plays an essential role in such a process, and identification of ical analysis of tissues derived from breast cancer patients shows molecules and characterization of the mechanisms regulating cell an increase of PLCg1 expression in metastasis compared with the motility is critical to understand metastasis development (1–4). primary tumor in around 50% of tissues analyzed. These data show Directed cell migration is regulated by chemokines and growth that PLCg1 is necessary for breast cancer–derived metastasis factors that bind and activate specific receptors, such as receptor development. Moreover, we show that inducible down-regulation of tyrosine kinases. Receptors convert these outside signals to activate PLCg1 expression after 14 days of injection of MDA-MB-231 in a complex network of intracellular pathways that regulate cell nude mice is able to revert metastasis formation, indicating that cytoskeleton rearrangement and motility. In this regard, activation blockade of PLCg1 has a therapeutic potential to counteract of the enzyme phospholipase Cg1 (PLCg1) is thought to play a metastasis dissemination.

Note: Supplementary data for this article are available at Cancer Research Online Materials and Methods (http://cancerres.aacrjournals.org/). Materials and constructs. Sequence targeting the human PLCg1 mRNA Current address for G. Sala: Laboratory of Molecular Oncology, Center of was subcloned into pSuper and pSuperior vectors (ref. 17; Supplementary Excellence on Aging, G. d’Annunzio University Foundation, Chieti, Italy. B Requests for reprints: Marco Falasca, Inositide Signalling Group, Centre for Fig. S1 ). Control vectors (pSuper 3Mut and pSuperior 3Mut) contain a Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Barts and three-point mutated sequence unable to target the human PLCg1 mRNA The London School of Medicine and Dentistry, Queen Mary, University of London, 4 (Supplementary Fig. S1B). Palm-HA-PLCg1 was kindly provided by Dr. E. Newark Street, London E1 2AT, United Kingdom. Phone: 44-0-20-7882-8243; Fax: 44-0- Bonvini (MacroGenics, Inc.). Antibodies were as follows: phosphorylated 20-7882-2186; E-mail: [email protected]. 783 I2008 American Association for Cancer Research. PLCg1(Tyr ), PLCg1, phosphorylated epidermal growth factor receptor doi:10.1158/0008-5472.CAN-08-1181 (EGFR; Tyr992), EGFR, phosphorylated AKT (Ser473), AKT, phosphorylated www.aacrjournals.org 10187 Cancer Res 2008; 68: (24). December 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research extracellular signal-regulated kinases (ERK), and ERKs from Cell Signaling; temperature and blocked with 0.1% bovine serum albumin for 30 min at phosphorylated focal adhesion kinase (FAK; Tyr397) from BioSource; Rac1, room temperature. Coverslips were then incubated for 2 h at room FAK, paxillin, and glyceraldehyde-3-phosphate dehydrogenase from Santa temperature with the appropriate primary antibody (antipaxillin, anti-FAK Cruz Biotechnology; phosphorylated Tyr (4G10) from UBI; and CD44 from antibodies). Rhodamine-labeled phalloidin was used to visualize actin Novocastra Laboratories Ltd. cytoskeleton. Confocal imaging was performed using a BIO-RAD Radiance Cell culture, transfection, and RNA interference. MDA-MB-231 and 2100 laser and an upright Nikon Eclipse E1000 microscope running U87 cells were grown in DMEM; MDA-MB-435 and TSA were grown in LaserSharp 2000 software. Ruffles formation was assessed as described (21). RPMI. Media were supplemented with 10% fetal bovine serum (FBS), Animals. Female nude athymic CD-1 nu/nu mice (7 wk old) and BALB/c sodium pyruvate, penicillin, and streptomycin and incubated in 5% CO2 at mice were purchased from Charles River Laboratories and maintained 37jC. TSA is an aggressive and poorly immunogenic cell line established under specific pathogen-free conditions with food and water provided ad from a moderately differentiated mammary adenocarcinoma that arose libitum. The general health status of the animals was monitored daily. spontaneously in a multiparous BALB/c mouse. Procedure involving animals and their care were conducted in conformity Down-regulation of PLCg1 was obtained by using pSuper retro and with the institutional guidelines that are in compliance with national and pSuperior retro–based vectors to express stable or inducible short hairpin international laws and policies. RNA (shRNA), respectively. Retroviral stocks were generated as described Experimental lung metastasis assay. Control (3Mut) and inducible/ (18). For the inducible system, down-regulation of PLCg1 expression was stable PLCg1 knockdown MDA-MB-231 cells were injected into the tail vein obtained by culturing cells in medium containing 2 Ag/mL doxycycline of recipient mice (2 Â 106 per mouse). Five weeks after cell implant/ (Sigma) or 10 Ag/mL tetracycline (Sigma). MDA-MB-231 stable cell lines injection, lung tissues (five lungs per group) were excised and immediately expressing the membrane-associated, palmitoylated HA-tagged PLCg1 were fixed in 10% neutral buffered formalin. Lungs were then embedded in transfected with 5 Ag of palmitoylated HA-tagged PLCg1 (Palm-PLCg-1-HA) paraffin, and thick sections (10 Am; two sections per lung tissue) were cut using Lipofectamine 2000 (Invitrogen) and then selected with 500 Ag/mL and stained with H&E or with an anti-CD44 monoclonal antibody from of G418. Santa Cruz Biotechnology. Rac1 activation assay. For EGF stimulation, 2 Â 106 cells plated in In vivo tumor growth. pSuperior 3Mut and shPLCg1 MDA-MB-231 cells 10-cm dishes were serum starved for 24 h and then stimulated with were injected s.c. into the left and right flanks of each mouse, respectively 20 ng/mL EGF (Sigma) for 5 min. For serum growing condition, 1 Â 106 cells (6 Â 106 per mouse). When tumors reached a size of f50 mm3, mice were were plated in 10-cm dishes and allowed to grow for 3 d in the absence or randomized in two groups (n = 5), with one group receiving tetracycline presence of doxycycline to down-regulate PLCg1 expression. Cells were (2 mg/mL; Sigma) in drinking water. Tetracycline was replaced every 2 d. then lysed on ice in lysis buffer [50 mmol/L Tris (pH 7.2), 100 mmol/L NaCl, The diameter of s.c. growing tumors was measured with a caliper twice a 5 mmol/L MgCl2, 1 mmol/L DTT, 10% glycerol, 1% NP40] containing week, and the experiments ended at day 35. The volume of s.c. growing protease inhibitors, and Rac GTP pull-down assays were then performed tumors was calculated by the formula: tumor volume (mm3) = (length Â using a GST-PAK-CRIB as described (19). width2) / 2. Differences in s.c. tumor growth, in the presence or in the Cell migration and invasion assay. Cell migration was performed absence of tetracycline, were evaluated with a one-way ANOVA followed by in Transwell chambers (tissue culture treated, 10-mm diameter, 8-Am Fisher’s test using the StatView statistical package (SAS Institute, Inc.). pores, Nunc) as described (20). Where indicated, inserts were coated with Differences were considered statistically significant at a level of P < 0.05. 10 Ag/mL fibronectin. Invasion assay in TSA and MDA-MB-435 cells was All data are expressed as mean F SE. performed in Matrigel-coated Transwell inserts (Matrigel diluted 1:8). Cells Mammary fat pad was performed in groups of eight mice receiving two (100,000 per transwell) were resuspended in RPMI containing 1% FBS and mammary fat pad inoculations consisting of 1 Â 105 TSA 3Mut and plated on top of the inserts, whereas RPMI containing 20% FBS was plated shPLCg1 cells resuspended in 50 AL media and mixed with equal volume of on the bottom. Cells were allowed to migrate for 48 h. Matrigel. Cells were implanted orthotopically into the left and right intact Three-dimensional collagen I invasion assay. Collagen I plugs fourth inguinal mammary fat pad of 6-wk-old to 8-wk-old female BALB/c (2 mg/mL) were prepared from fibrillar bovine collagen I (3.1 mg/mL; mice. Tumor growth was measured daily with calipers. PureCol) by dilution in DMEM in accordance with the manufacturer’s Western blot of mice tissues. Tumor samples were collected, snap protocol (Nutacon). A collagen solution (2.5 mL) was placed in a 24-mm frozen, and analyzed by Western blot. Frozen specimens of tumor tissue diameter, 8-Am pore transwell insert (Corning) and allowed to polymerize were homogenized with a Polytron homogenizer in a lysis buffer (ratio, 1:1 for 60 min at 37jC. Cells (2.5 Â 104) were plated in 1.5 mL of serum-free w/v) containing 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, 0.1% NP40, DMEM on top of the plug. To establish a chemoattractant gradient, 2.5 mL 250 mmol/L NaCl, 50 mmol/L NaF in the presence of leupeptine, pepstatine, of DMEM containing 20% FCS were added to the lower chamber and aprotinin, and phenyl-methyl-sulfonyl-fluoride as proteases inhibitors. The replaced every 10 to 12 h to maintain the gradient. For experiment with homogenates were then centrifuged at 13,000 rpm for 10 min at 4jC, and inducible PLCg1 knockdown, cells were treated with doxycycline 48 h the protein concentration was determined using a Bio-Rad assay kit before plating. Cells were allowed to invade for 5 d, then fixed in 4% (Bio-Rad). Eighty micrograms of proteins were separated on SDS-PAGE and paraformaldehyde, washed thrice in PBS, permeabilized with 0.2% Triton transferred to a polyvinylidene difluoride membrane (Millipore). The X-100, and stained with Sytox green (Invitrogen, 1:5,000). Confocal imaging membrane was probed with the indicated primary antibodies, washed was performed using a BIO-RAD Radiance 2100 laser and an upright Nikon with TBS-T (TBS 1Â + 0.05% Tween 20), and then incubated for 1 h with Eclipse E1000 microscope (10 Â LWD objective) running LaserSharp 2000 horseradish peroxidase–conjugated secondary antibody in TBB buffer software. Analysis was performed by taking sections at 6-Am intervals (TBS-T + 5% nonfat milk). Proteins were detected by enhanced chemilu- through 400 to 900 Am of the collagen plug. Cells that had invaded >100 Am minescence (Amersham). were considered as invading cells. The percentage of invading cells was Immunohistochemistry analysis. Expression of PLCg1 was analyzed in determined by dividing the sum of the fluorescence in invading sections formalin-fixed, paraffin-embedded human breast tumors and matching by the total fluorescence of all sections. Levels of fluorescence were metastasis (n, 60 pairs), using an anti-PLCg1 monoclonal antibody from determined by Image J. Three-dimensional reconstructions were performed Santa Cruz Biotechnology. Briefly, antigen retrieval was performed by using LSM Browser (Zeiss) imaging software. heating for 15 min in 10 mmol/L sodium citrate buffer (pH 6.0; DAKO, Cell proliferation assay. 3Mut and shPLCg1 MDA-MB-231 cells S2031) in a microwave oven. The slides were incubated overnight at 4jC (2 Â 104) were seeded on six-well plates in DMEM containing 10% FBS. with a 1:150 dilution of the primary antibody, and the reaction was Proliferation was assessed at different days by using the cell counter CDA- evidenced with Dako EnVision kit. Diaminobenzidine was used as the 500 (Sysmex). chromogen, and the slides were counterstained with Mayer hematoxylin. In Confocal microscopy and ruffling assay. Cells were treated as control sections, the specific primary antibody was omitted or replaced indicated, then fixed in 4% paraformaldehyde for 15 min at room with nonimmune serum or isotype-matched immunoglobulins (Dako). The

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Phospholipase C; in Breast Cancer Metastasis staining of at least 500 cells in primary tumors and metastatic lesions was mutation (7Mut), which prevent the shRNA targeting of PLCg1 evaluated by two independent pathologists (MP, RL) using a modified mRNA (Supplementary Fig. S1B). As both mutated shRNA PLCg1 histochemical score (H-score; ref. 22) The H-score includes an assessment of failed to down-regulate PLCg1 expression (Supplementary Fig. both the intensity of staining and the percentage of stained cells. For the S1A), we elected the 3Mut sequence as the best appropriate control intensity, a score index of 0, 1, 2, and 3 corresponding to negative, weak, for all further experiments. PLCg1 knockdown (ranging from 90% moderate, and strong staining intensity, respectively, was used, and the percentage of positive cells at each intensity was estimated. Disagreement to 100% of control) had no effect on EGFR phosphorylation C between the pathologists was noted for <8% of samples. In these cases, (Supplementary Fig. S1 ) and it was isoform-specific because D consensus was reached by a joint reevaluation of the slide. A final score of PLCg2 expression was unaffected (Supplementary Fig. S1 ). For 0 to 300 is the product of both the intensity and the percentage. An H-score the inducible PLCg1 knockdown, one selected tetracycline- difference of at least 25% between breast tumors and matching metastasis inducible MDA-MB-231 clone was infected with the pSuperior was considered indicative of a different PLCg1 expression. shPLCg1 and pSuperior 3Mut retro vectors. Addition of tetracycline-induced PLCg1 knockdown in pSuperior shPLCg1 but not in pSuperior 3Mut MDA-MB-231 cells (Supplementary Fig. S1E). Results PLC;1 regulates cytoskeletal rearrangements and Rac1 Modulation of PLC;1 levels in breast cancer cell lines. To activation. Several lines of evidence indicate that the PLCg1 study the role of PLCg1 in cell metastasis, we analyzed the effect of substrate PtdIns-4,5-P2 (24) and the enzyme itself (25) are involved both stable and inducible down-regulation of PLCg1 expression in cytoskeletal rearrangement through modulation of actin-binding obtained by RNA interference (RNAi) strategy. As a complementary proteins. Therefore, we analyzed the actin cytoskeleton in control approach, we tested the effect of overexpression of Palm-PLCg1- (3Mut) and PLCg1 knockdown MDA-MB-231 cells in the presence HA (23). of serum. We observed that both stable (data not shown) and Specific shRNA targeting PLCg1 (shPLCg1) were subcloned into inducible (pSuperior; Fig. 1A) PLCg1 down-regulation induced a a pSuper retro or pSuperior retro–based vectors to obtain a stable marked decrease in the number of membrane ruffles. To further or inducible down-regulation of PLCg1 expression, respectively investigate the effect of PLCg1 knockdown in a distinct breast (Supplementary Fig. S1). As a control, we generated cells expressing cancer cell line, we infected the murine mammary adenocarcinoma shRNA PLCg1 carrying a three-point mutation (3Mut) or a 7-point cells TSA with 3Mut and PLCg1 shRNA (PLCg1 down-regulation is

Figure 1. PLCg1 regulates membrane ruffles formation and Rac activity in MDA-MB-231 cells. A, pSuperior 3Mut and shPLCg1 MDA-MB-231 cells were kept in serum, treated with doxycycline for 48 h, and then fixed. Membrane ruffles formation was analyzed by confocal microscopy using a Rhodamine-labeled phalloidin antibody to visualize actin cytoskeleton. Bar, 10 Am. B, serum-starved TSAs were stimulated with serum or EGF for 6 min, fixed, and then analyzed by confocal microscopy. The number of cells showing membrane ruffles was assessed, as described (21). Data are mean F SE of three independent experiments. C, the indicated cells were serum starved for 24 h and then unstimulated (sf) or stimulated with 20 ng/mL EGF (EGF) for 5 min. Alternatively, cells were kept in serum for 48 h (serum). Pull-down assay to detect GTP-bound Rac was performed, as described in Materials and Methods. Down-regulation of PLCg1 levels was assessed by using a specific antibody. D, densitometry analysis of Rac activation. Columns, mean of three independent experiments; bars, SE. www.aacrjournals.org 10189 Cancer Res 2008; 68: (24). December 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research shown below). Serum-starved TSA were stimulated with serum or impaired both the EGF-induced and serum-induced Rac1 activa- EGF, and the percentage of cells showing ruffles was determined. tion (Fig. 1C and D). Similar data were obtained in stable Data revealed that both serum-induced and EGF-induced mem- knockdown cells (data not shown). The EGF-induced activation brane ruffles formation was clearly reduced in PLCg1 knockdown of Akt was unaffected by PLCg1 down-regulation, whereas a cells (Fig. 1B). reduction in the late phase of ERK activation was detected Because it is well established that the small GTP binding protein (Supplementary Fig. S2). Rac is directly involved in membrane ruffles formation (26), we To further assess the role of PLCg1 in this process, we analyzed then analyzed the activation status of Rac in MDA-MB-231 cells. As the effect of overexpression of a palmitoylated PLCg1 mutant shown in Fig. 1C and D, both serum and EGF were able to activate which is constitutively associated to the plasma membrane (Palm- Rac1 in control 3Mut MDA-MB-231 cells. Similarly, serum was able PLCg1-HA) and has been shown to act as a constitutively active to activate Rac1 in HEK293T cells used as control (Fig. 1C). On PLCg1 mutant (23). Overexpression of Palm-PLCg1-HA induced a the contrary, inducible down-regulation of PLCg1 significantly marked increase in membrane ruffles formation in serum

Figure 2. Down-regulation of PLCg1 expression impairs cell invasion. Collagen assay in A and B was performed, as described in Materials and Methods. A and B, representative images of MDA-MB-231 (A) and U87 (B) within and on topof the collagen plug.Cells were seeded at the topof the filter and allowed to migrate for 5 d toward the chemoattractant (bottom). N and I indicate noninvading and invading cells. Quantitative analysis of invasion are also shown. Columns, mean of three independent experiments; bars, SD. C and D, serum-induced invasion of pSuper 3Mut and shPLCg1 TSA (C) and MDA-MB-435 (D) on Matrigel-coated Transwell inserts. Columns, mean of three (C) and four (D) independent experiments; bars, SE. *, P < 0.05.

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Figure 3. PLCg1 expression is required for focal adhesion formation. A, pSuper 3Mut and shPLCg1 MDA-MB-231 were seeded on glass coverslips coated with 20 Ag/mL laminin and allowed to adhere for 20 min before fixing for confocal microscopy analysis. Focal adhesions were visualized by using an anti-FAK antibody. Actin was visualized by using rhodamine phalloidin. B, pSuperior 3Mut (a, b, c) and shPLCg1(d, e, f) cells were grown in the presence of 2 Ag/mL doxycycline for 48 h, seeded on glass coverslips coated with 10 Ag/mL fibronectin, and allowed to adhere for 60 min before fixing for confocal microscopy analysis. Arrows indicate focal adhesions visualized by using an anti-paxillin antibody. C, pSuper 3Mut and shPLCg1 MDA-MB-231 were plated on laminin or fibronectin-coated well. After 20 min, cells were lysed and analyzed by Western blotting as indicated. Cells left in suspension (susp) were used as control.

(Supplementary Fig. S3A) and an increase in Rac activation Furthermore, a clear inhibition of invasion on Matrigel-coated (Supplementary Fig. S3B) in both serum-stimulated and EGF- transwell inserts was observed in TSA (Fig. 2C) and in human stimulated MDA-MB-231 compared with cells expressing the empty breast cancer cell line MDA-MB-435 upon PLCg1 down-regulation vector pcDNA3. PLCg1 overexpression is shown in Supplementary (Fig. 2D). Efficiency of PLCg1 knockdown in MDA-MB-435 and U87 Fig. S3C. Taken together, these data indicate that PLCg1 is required cells is shown in Supplementary Fig. S5A and B, respectively. Taken for Rac1 activation and membrane ruffles formation. together, these data show that PLCg1 is necessary for cell RNAi-mediated inhibition of PLC;1 expression impairs cell migration and invasion in vitro. migration, invasion, and cell spreading in vitro. The observa- A random motility assay in MDA-MB-231 cells overexpressing tion that PLCg1 was able to modulate both Rac1 activation and Palm-PLCg1-HA revealed a defect in migration (data not shown). cytoskeletal rearrangements strongly suggested that this enzyme Indeed, these cells showed intense formation of protrusions all might have a role in cell migration. This hypothesis was further around the cells, possibly due to the PLCg1 localization and supported by our previous work reporting a role for a PI3K/PLCg1 subsequent Rac1 activation all around the perimeter of the cells. pathway in migration of MDA-MB-231 cells (15). Migration assays This may affect cell polarization, ultimately resulting in the in control and PLCg1 knockdown MDA-MB-231 cells revealed that observed impairment in cell migration. PLCg1 down-regulation clearly reduced EGF-induced cell migra- To gain further insight into the mechanisms of the PLCg1- tion (Supplementary Fig. S4A) and basal cell migration on dependent cell migration and invasion, we performed spreading fibronectin (Supplementary Fig. S4B). Moreover tridimensional assays on cells plated on laminin and fibronectin. Both stable invasion assays showed that inducible (Fig. 2A) or stable (data not (Fig. 3A) and inducible (Fig. 3B) PLCg1 knockdown cells seem shown) PLCg1 knockdown resulted in a complete impairment of round and less spread than control (3Mut) cells. In addition, cell invasion. Similar data were obtained by down-regulating staining with antibodies to focal adhesion kinase (FAK; Fig. 3A) and PLCg1 expression in U87 (Fig. 2B), a glioblastoma cell line whose paxillin (Fig. 3B) clearly revealed a decrease in focal adhesion cell migration has been shown to be PLC-dependent (27). number in PLCg1 knockdown cells. Parallel biochemical analysis www.aacrjournals.org 10191 Cancer Res 2008; 68: (24). December 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research showed that neither FAK nor Akt activation (assessed by moni- experimental condition, MDA-MB-231 cells were still expressing toring phosphorylation of their residues Tyr397 and Ser473, respec- PLCg1 and, indeed, were able to develop lung metastases after tively) were impaired in these experimental conditions (Fig. 3C). 14 days from injection (Fig. 5A). After 14 days, animals were divided On the contrary, a clear decrease of ERK phosphorylation was in two groups, one untreated and one treated with tetracycline, observed in PLCg1 knockdown MDA-MB-231 cells plated on to down-regulate PLCg1 levels. Interestingly, we observed that laminin and fibronectin (Fig. 3B). These data indicate that PLCg1 reduction of PLCg1 expression was able to induce regression of down-regulation impairs spreading on laminin and fibronectin of metastasis formation. Specifically, at the end point of the MDA-MB-231 possibly through impairment of ERK activation. experiment (60 days), we found that four of five mice kept in PLC;1 is necessary for breast cancer cell–derived lung the absence of tetracycline (control) showed lung metastases metastasis in vivo. Data, thus far, revealed that PLCg1 is required (Fig. 5B). On the contrary, after day 14, five of six mice treated for cell migration and invasion. Because these processes are key with tetracycline showed completely metastasis-free lungs, whereas steps in metastasis development, we decided to investigate the only the remaining single mouse presented few, barely detect- effect of PLCg1 knockdown in an in vivo experimental metastasis able metastases (Fig. 5B). These data indicate that not only PLCg1 assay. As shown in Fig. 4, control MDA-MB-231 (3Mut) cells has a key role in metastasis formation but also that blockade of injected in mice tail vein induced formation of lung metastases in PLCg1 reverts metastasis formation and, therefore, could have 4 to 5 weeks. Strikingly, when we injected stable (Fig. 4A)or therapeutic potential. inducible (Fig. 4B) PLCg1 knockdown MDA-MB-231 cells, metas- PLC;1 expression is increased in lymph node metastasis tasis formation was almost completely abrogated. In fact, four of compared with primary tumors in breast cancer patients. five lung tissues in both stable and inducible PLCg1 knockdown Data, thus far, strongly indicate that PLCg1 has a role in breast were completely metastasis-free and only one sample showed few cancer–derived lung metastasis development. To further assess the metastases. Lung sections (stable; Fig. 4A) or external lung pictures involvement of this enzyme in such a process, we performed (inducible; Fig. 4B) from this single mouse bearing few metastases immunohistochemical analysis of samples from 60 breast cancer are shown. patients to compare the expression levels of PLCg1 in lymph node Because PLCg1 may be involved in different steps involved metastasis to primary tumors. Two of 60 primary tumors did not in metastasis formation, we decided to investigate whether express PLCg1, whereas all metastases showed specific PLCg1 it was possible to block and possibly revert metastasis by down- staining. Representative tissues are shown in Fig. 5C. We found regulating PLCg1 expression after metastasis had already devel- that PLCg1 expression is significantly increased in lymph node oped. To this purpose, we injected pSuperior shPLCg1 cells and metastasis compared with primary tumors (Wilcoxon matched- kept the injected mice in the absence of tetracycline. In this pairs signed ranks test, W+ = 412.50, WÀ = 913.50, n = 51,

Figure 4. PLCg1 is required for MDA-MB-231 cell–derived lung metastasis in vivo. A, control (3Mut) and stable shPLCg1 cells were injected in the tail vein of nude mice. Experimental metastasis assay was performed, as described in Materials and Methods. Two representative images of lung sections stained with hematoxilin and eosin are shown. B, control (3Mut) and inducible shPLCg1 cells were injected in the tail vein of nude mice. External lung pictures are shown. Bottom, Western blotting analysis of lysates from pSuperior 3Mut and shPLCg1 cells after 48 h of treatment with tetracycline and before the injection.

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Figure 5. PLCg1 is important for metastasis progression. A and B, pSuperior shPLCg1 MDA-MB-231 cells (1 Â 106) were injected in nude mice. Mice were kept in the absence of tetracycline to maintain PLCg1 expression. After 14 d, three animals were sacrificed and the presence of micrometastasis in lung sections was analyzed by CD44 staining (A). The remaining mice were divided in two groups and treated (n = 6) or not (n = 6) with tetracycline to down-regulate PLCg1 expression. Mice were sacrificed after 46 d (60 d from implant). Representative lung sections from untreated mice or mice treated with tetracycline are shown. Arrows indicate micrometastasis. C, expression of PLCg1 in three primary breast tumors and matching lymph node metastases assessed by immunohistochemistry. Corresponding control images are shown in insets.

P < 0.01911). Data revealed that, in 48% of breast cancer patients, Discussion PLCg1 expression was markedly increased in lymph node Here, we show that PLCg1 is a critical enzyme in the metastases compared with the corresponding primary tumors, development and maintenance of tumor metastasis. Our data further supporting the role for this enzyme in metastases provide evidence that dissemination of tumor cells in models of development. breast cancer, as well as in the human disease, correlates with ; Effects of PLC 1 down-regulation on primary tumor PLCg1 expression. In addition our data indicate that loss of PLCg1 growth. To test the effect of PLCg1 down-regulation on primary affects actin polymerization and focal adhesion formation through in vitro in vivo tumor growth, we studied and growth of pSuperior modulation of the Rho family GTPase Rac. Interestingly, our in vitro shPLCg1 MDA-MB-231 cells. No difference in growth, data not only reveal that PLCg1 is necessary for metastasis assessed by cell counting, was observed in inducible PLCg1 formation but they also show that down-regulation of the enzyme knockdown MDA-MB-231 cells compared with the control (3Mut) expression results in metastasis regression. A cells upon 5 days of culture (Fig. 6 ). We then implanted Taken together, our findings suggest that metastatic dissemina- inducible PLCg1 knockdown MDA-MB-231 cells s.c. in nude mice tion can be controlled by targeting PLCg1 and its downstream and tested their growth in vivo in the presence or absence of signaling pathways. tetracycline. A slight, although not significant, difference in Tumor metastasis is a complex process involving different primary tumor growth was observed in mice implanted with sequential steps (1, 3, 4, 28), of which cell motility and invasion are pSuperior shPLCg1 MDA-MB-231 compared with control (3Mut)- critical events. Several chemoattractants can activate cell migration injected mice (Fig. 6B). In vivo tumor growth of pSuperior 3Mut and invasion through activation of their specific receptors and was unaffected by tetracycline treatment (data not shown). downstream intracellular signaling pathways, ultimately resulting Efficiency of PLCg1 down-regulation was verified in cells tested in reorganization of the actin cytoskeleton. Several components of in in vitro growth (Fig. 6A) and in excised tumors (Fig. 6B). To such signaling pathways have been identified and found overex- better analyze the effect of PLCg1 down-regulation on primary pressed in invasive tumor cells. Among these Rho/Rac GTPases are tumor growth, we implanted TSA cells in the mammary fat pad. well-known regulators of cytoskeletal organization (2, 6). In Data revealed that tumor growth was slightly, but significantly, particular Rac activation induces membrane ruffles, focal adhesion reduced in mice implanted with PLCg1 knockdown cells complexes, and lamellipodia formation (2, 26). The latter promotes compared with controls (Fig. 6C). cells to extend protrusions requested for cell polarity and forward www.aacrjournals.org 10193 Cancer Res 2008; 68: (24). December 15, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research movement. The intracellular mechanism regulating Rac1 activation induces translocation of PLCg1 to the leading edge of MDA-MB- in cell migration is not completely understood. PI3Ks together with 231 migrating cells (15), our present data suggest that PLCg1may Rac seem to be key regulators of both polarization and cell be the intracellular link between PI3K and Rac activity. migration (26), and several evidences suggest the existence of a Given its essential role in cell motility Rac has been implicated in mutual regulation of PI3K and Rac activity although its precise cancer cell invasion and metastasis. A direct correlation between nature is, at present, unclear. Here, we show that PLCg1 regulates Rac activation and metastatic potential of a panel of cell variants Rac1 activation in breast cancer cells. Because we have shown that derived from MDA-MB-435 breast cancer cell line has been lipid product of PI3K contribute to PLCg1 activation (29) and reported (30). In addition RNAi-mediated knockdown of Rac

Figure 6. In vitro and in vivo tumor growth. A, pSuperior 3Mut and shPLCg1 MDA-MB-231 cells treated with 2 Ag/mL doxycycline were seeded and cultured in the presence of serum. At the indicated times, cells were trypsinized and counted. PLCg1 knockdown in these cells is also shown. B, exponentially growing pSuperior shPLCg1 MDA-MB-231 cells, treated or not with tetracycline, were injected into the right flank and the left flank, respectively, of the recipient mice. Tumor growth was assessed, as described in Materials and Methods. Western blotting analysis and corresponding densitometry analysis (bottom) of homogenates from tumors excised at the end of the experiments. C, 3Mut and shPLCg1 TSA cells were implanted in the mammary fat pad, and tumor growth was assessed as indicated. *, P < 0.05; **, P < 0.01. PLCg1 levels in pSuper 3Mut and shPLCg1 TSA cells are also shown. Equal loading was confirmed by using an anti-Akt antibody.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Phospholipase C; in Breast Cancer Metastasis strongly inhibits lamellipodia formation and cell migration of signaling correlated with a decreased breast cancer cell prolifer- SNB19 glioblastoma cell line and BT549 breast cancer cell line (31). ation rate (35). Our data suggest that PLCg1 regulates both cell Among the different Rac downstream effectors, Wiskott-Aldrich migration and, possibly, metastasis proliferation. Interestingly, this syndrome protein (WASP) family proteins seems to have an effect seems to be metastasis-specific because the primary tumor important role in cell migration and metastasis (32). Therefore, growth is only slightly affected by PLCg1down-regulation. PLCg1 may regulate actin cytoskeleton reorganization through Moreover, this effect is significant only when cells are injected in Rac and WASP family proteins. However, we cannot rule out the the mammary fat pad. One explanation would be that PLCg1 possibility that PLCg1 can regulate cell migration through signaling might have a key role on the cell proliferation of a downstream effectors other than Rac. In this respect, it is subcellular population of cancer cells critical for metastasis noteworthy that PLCg1 directly activates cofilin (33). Increasing development or that PLCg1 controls a signaling pathway that evidences indicate that cofilin is an essential regulator of cancer is more important for metastatic growth than for primary motility and invasion and that its activity is regulated by a PLC tumor growth. This is further supported by our data obtained in (33, 34). A role for PLCg1 in cofilin activation is supported by breast cancer patients and the recent data indicating that breast our observations that PLCg1 down-regulation induces changes in cancer metastases are molecularly distinct from their primary GFP-cofilin intracellular localization in knockdown MDA-MB-231 tumors (36). cells compared with their respective control.4 Another intriguing Alternatively, because our data indicate that PLCg1 is involved in possibility is that PLCg1 can regulate cell migration and invasion focal adhesion formation and cell interaction with microenviron- through modulation of ERK activation. Indeed several lines of ment, a lack of such interactions in the absence of PLCg1may evidence indicate that ERK is involved in cell migration, invasion, explain the observed regression of micrometastasis after inducible and metastasis development (28). These data, together with our knockdown of PLCg1. This hypothesis is supported by recent data observation that down-regulation of PLCg1 expression impairs a showing that small interfering RNA knockdown of PLCg1in late-stage EGF-induced ERK activation (Supplementary Fig. S2) human prostate carcinoma cell line (PC3LN3) induces defect in cell and ERK activation in cells plated on laminin or fibronectin (Fig. adhesion (37). 3B), strongly suggest that ERK may be another downstream effector In conclusion, our data strongly suggest that PLCg1 inhibitors of PLCg1 in regulating those processes. may have potential therapeutic applications for the clinical Our data clearly indicate that PLCg1 plays a key role in treatment of tumor metastasis. At present, no specific PLCg1 metastasis formation. Indeed both stable and inducible PLCg1 inhibitors are available, and therefore, there is a stringent need to down-regulation results in an almost complete inhibition of breast develop novel-specific PLCg1 inhibitors. cancer–derived lung metastasis formation. Previous work using a dominant-negative PLCg1 had shown a partial inhibition of Disclosure of Potential Conflicts of Interest metastasis formation in a model of spontaneous prostate No potential conflicts of interest were disclosed. carcinoma (16), although it should be noticed that the use of this construct may induce unspecific effects. On the contrary, our strategy was based on modulating PLCg1 levels by both stable Acknowledgments and inducible RNAi-dependent down-regulation. We further show Received 3/31/2008; revised 9/17/2008; accepted 9/21/2008. g Grant support: Association for International Cancer Research grant 05-127 that down-regulation of PLC 1 expression is able to induce (M. Falasca), European Commission FP6 Program Apotherapy EC contract 037344 metastasis regression. (http://apotherapy.med.uoc.gr; M. Falasca and M. Broggini), and Fondazione These data suggest that PLCg1 is important not only in the early Carichieti. G. Sala was supported by Association for International Cancer Research grant 05-127 (M. Falasca). steps of the metastatic cascade, such as cell migration and The costs of publication of this article were defrayed in part by the payment of page invasion, but also at a late stage in metastasis development. charges. This article must therefore be hereby marked advertisement in accordance Previous work has already shown that inhibition of PLCg1 with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Prof. J.F. Martin and Prof. P. Innocenti for support, Dr. Ezio Bonvini for providing Palm-PLC;1-HA, Dr. G. Baldanzi for help with invasion assays, R. La Sorda for help in immunohistochemical analysis, and Dr. P. Frankel, Prof. I. Zachary, Dr. S. 4 G. Sala and M. Falasca, unpublished observation. Anastasi, Dr. A. Sala, and Dr. O. Segatto for advice and reagents.

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Gianluca Sala, Francesco Dituri, Claudio Raimondi, et al.

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